Sample preparation methods for maldi mass spectrometry

Abstract

This invention provides methods for preparing samples for MALDI mass spectrometry. The methods make possible the use of reagents that are normally considered unsuitable for MALDI in the preparation of a sample. Also provided are methods for internal calibration of a mass spectrometer, and methods for preparing sample supports.

What is claimed is:  
     
         1 . A method for preparing a sample for matrix-assisted laser desorption/ionization (MALDI) spectrometry, the method comprising: 
 spotting onto a MALDI support an aliquot of a matrix;    depositing an analyte onto the MALDI support at a same position as the aliquot of the matrix, wherein either the aliquot of matrix or the analyte further comprises a MALDI-incompatible solvent;    allowing the matrix and analyte to dry onto the MALDI support to form a cocrystallizate;    depositing onto the co-crystallizate a recrystallization solution into which the cocrystallizate redissolves; and    allowing the redissolved co-crystallizate to dry, thereby forming a sample suitable for MALDI.    
     
     
         2 . The method of  claim 1 , wherein the matrix comprises a component selected from the group consisting of α-cyano-4-hydroxycinnamic acid, sinapic acid, 2-(4-hydroxyphenylazo) benzoic acid, succinic acid, 2,6-dihydroxyacetophenone, ferulic acid, caffeic acid, glycerol, 4-nitroaniline, 2,4,6-trihydroxyacetophenone, 3-hydroxypicolinic acid, anthranilic acid, nicotinic acid, salicylamide, trans-3-indoleacrylic acid, dithranol, 2,5-dihydroxybenzoic acid, 3,5-dihydroxybenzoic acid, isovanillin, 3-aminoquinoline, T-2-(3-(4-t-butyl-phenyl)2-methyl-2-propenylidene)malanonitrile, and 1-isoquinolinol.  
     
     
         3 . The method of  claim 2 , wherein the matrix comprises a polymer, oligomer, or self-assembled monolayer of one or more of the components.  
     
     
         4 . The method of  claim 1 , wherein the MALDI-incompatible solvent comprises a chaotropic agent.  
     
     
         5 . The method of  claim 1 , wherein the MALDI-incompatible solvent comprises a solvent having a low vapor pressure.  
     
     
         6 . The method of  claim 1 , wherein the MALDI-incompatible solvent comprises dimethylformamide, dimethylsulfoxide, N-methylpyrrolidone, methylene chloride, polyethylene glycol, glycol or glycerol.  
     
     
         7 . The method of  claim 1 , wherein the matrix is a hydrophobic matrix and the MALDI-incompatible solvent is one that can solvate the matrix.  
     
     
         8 . The method of  claim 7 , wherein the matrix is α-cyano-4-hydroxycinnamic acid or sinapic acid and the MALDI-incompatible solvent comprises 100% dimethylformamide.  
     
     
         9 . The method of  claim 1 , wherein the matrix is a hydrophilic matrix and the MALDI-incompatible solvent is one that can solvate the analyte.  
     
     
         10 . The method of  claim 1 , wherein the MALDI-incompatible solvent comprises a sample solubilization agent.  
     
     
         11 . The method of  claim 10 , wherein the sample solubilization agent comprises one or more compounds selected from the group consisting of urea, a surfactant, and a salt.  
     
     
         12 . The method of  claim 1 , wherein the MALDI support comprises hydrophilic target regions upon which the aliquots of the matrix are spotted.  
     
     
         13 . The method of  claim 1 , wherein spotting the aliquot of the matrix onto the MALDI support is performed prior to depositing the analyte onto the MALDI support.  
     
     
         14 . The method of  claim 1 , wherein depositing the analyte onto the MALDI support is performed prior to spotting the aliquot of the matrix onto the MALDI support.  
     
     
         15 . The method of  claim 1 , wherein the analyte and the aliquot of the matrix are co-deposited onto the MALDI support.  
     
     
         16 . The method of  claim 1 , wherein the recrystallization solution comprises a MALDI-compatible solvent.  
     
     
         17 . The method of  claim 16 , wherein the MALDI-compatible solvent comprises one or more of water, acetonitrile (ACN), acetone, ethanol, methanol, trifluoroacetic acid (TFA), and formic acid.  
     
     
         18 . The method of  claim 17 , wherein the MALDI-compatible solvent comprises a solution of 75% ACN, 24.9% water and 0.1% TFA by volume.  
     
     
         19 . The method of  claim 16 , wherein the recrystallization solution further comprises one or more matrix components, a performance enhancing agent, or a combination thereof.  
     
     
         20 . The method of  claim 1 , wherein the method further comprises: 
 washing the MALDI support prior to depositing the recrystallization solution onto co-crystallizate.    
     
     
         21 . The method of  claim 1 , wherein a volume of the recrystallization solution deposited onto the co-crystallizate is less than a volume of the aliquot of matrix and MALDI-incompatible solvent.  
     
     
         22 . The method of  claim 21 , wherein the volume of the recrystallization solution deposited onto the co-crystallizate is less than 10 μL.  
     
     
         23 . The method of  claim 21 , wherein the volume of the recrystallization solution deposited onto the co-crystallizate is less than 5 μL.  
     
     
         24 . The method of  claim 21 , wherein the volume of the recrystallization solution deposited onto the co-crystallizate is less than 1 μL.  
     
     
         25 . The method of  claim 21 , wherein the volume of the recrystallization solution deposited onto the co-crystallizate is less than 0.5 μL.  
     
     
         26 . The method of  claim 21 , wherein the volume of the recrystallization solution deposited onto the co-crystallizate is less than 100 nL.  
     
     
         27 . The method of  claim 1 , wherein the method further comprises repeating one or more times the steps of depositing the recrystallization solution and allowing the redissolved cocrystallizate to dry.  
     
     
         28 . A sample on a sample support for MALDI mass spectrometry as prepared by the method of  claim 1 .  
     
     
         29 . A method for internal calibration of mass data generated by a mass spectrometer, the method comprising: 
 a) providing a support that comprises an analyte at a first location on the support, and a calibrant at a second location on the support;    b) ionizing the analyte and transiently storing analyte ions in an ion storage chamber;    c) ionizing the calibrant and transiently storing calibrant ions in the ion storage chamber; and    d) releasing a mixture of analyte ions and calibrant ions from the ion storage chamber into a mass analyzer.    
     
     
         30 . The method of  claim 29 , wherein step b) is performed prior to step c).  
     
     
         31 . The method of  claim 29 , wherein step c) is performed prior to step b).  
     
     
         32 . The method of  claim 29 , wherein the second location on the support comprises one or more side regions of the support.  
     
     
         33 . The method of  claim 29 , wherein transiently storing the analyte ions and the calibrant ions comprises trapping the ions with one or more multipole ion guides, trap electrodes, Penning traps, or a combination thereof.  
     
     
         34 . The method of  claim 33 , wherein the one or more multipole ion guides comprise a quadrupole ion guide, a hexapole ion guide, a octopole ion guide, a stacked ring ion guide, or a combination thereof.  
     
     
         35 . The method of  claim 29 , wherein the ion storage chamber comprises an ion trap that provides for mass selection.  
     
     
         36 . The method of  claim 35 , wherein mass selection is performed during ion transport into the ion storage chamber.  
     
     
         37 . The method of  claim 29 , wherein transiently storing the analyte ions and the calibrant ions comprises guiding the ions using one or more ion optics elements to an entrance of a first mass analyzer, and passing the ions through the first mass analyzer into the ion storage chamber.  
     
     
         38 . The method of  claim 37 , wherein passing the analyte ions or the calibrant ions through the first mass analyzer further comprises performing mass selection on the ions.  
     
     
         39 . The method of  claim 29 , wherein providing the support comprises placing the support on a movable x-y-stage; and wherein ionizing the analyte and ionizing the calibrant comprises moving the support to sequentially position the analyte and the calibrant in line with a laser beam.  
     
     
         40 . The method of  claim 29 , wherein ionizing the analyte and ionizing the calibrant comprises sequentially moving a laser beam in line with the analyte at the first position and the calibrant at the second position.  
     
     
         41 . A method for internal calibration of a mass spectrometer, the method comprising: 
 a) ionizing an analyte by a first method of ionization, transporting the resulting analyte ions into an ion storage chamber through a first set of ion optical elements, and transiently storing the analyte ions in the ion storage chamber;    b) ionizing a calibrant by a second method of ionization, transporting the resulting calibrant ions into an ion storage chamber through a second set of ion optical elements, and transiently storing the calibrant ions in the ion storage chamber; and    c) releasing the mixture of calibrant and analyte ions into a mass analyzer.    
     
     
         42 . The method of  claim 41 , wherein step a) is performed prior to step b).  
     
     
         43 . The method of  claim 41 , wherein step b) is performed prior to step a).  
     
     
         44 . The method of  claim 41 , wherein ionizing the analyte and ionizing the calibrant comprise performing ESI.  
     
     
         45 . The method of  claim 41 , wherein ionizing the analyte and ionizing the calibrant comprise performing MALDI.  
     
     
         46 . The method of  claim 41 , wherein ionizing the analyte and ionizing the calibrant comprise performing different ionization techniques.  
     
     
         47 . The method of  claim 41 , wherein the first and second sets of ion optical elements comprise a same set of ion optical elements.  
     
     
         48 . The method of  claim 41 , wherein the ion storage chamber comprises an ionization region of an ion source.  
     
     
         49 . The method of  claim 41 , wherein the mass spectrometer comprises a FT-ICR mass spectrometer.  
     
     
         50 . The method of  claim 41 , wherein ionizing the analyte and ionizing the calibrant comprises positioning an ion source proximal to the first or second set of ion optical elements.  
     
     
         51 . A method for making a sample support having a hydrophobic surface having one or more hydrophilic target regions, the method comprising: 
 providing a solid support comprising a hydrophobic surface;    positioning a mask on the hydrophobic surface, wherein the mask comprises one or more openings that are positioned at desired locations of the hydrophilic target regions;    placing the solid support and the mask under reduced air pressure; and    contacting the desired locations of the hydrophilic target regions with a plasma, wherein the plasma renders the hydrophobic surface under the one or more openings in the mask hydrophilic by reactive ion etching, thereby creating the hydrophilic target regions on the hydrophobic surface.    
     
     
         52 . The method of  claim 51 , wherein providing the solid support comprises exposing the surface to a plasma comprising one or more fluoro compounds.  
     
     
         53 . The method of  claim 51 , wherein the hydrophobic surface comprises PTFE, PTE, PE, PFA, graphite, a monolayer prepared from 1H, 1H, 2H, 2H perfluorodecyltrichlorosilane, a monolayer of octadecyltrichlorosilane, perfluoro alkylate, perfluoromethacrylate, polysilane, polysiloxane, fluoroalkyl-substituted polysilane., or fluoroalkyl-substituted siloxane.  
     
     
         54 . The method of  claim 51 , wherein the plasma comprises a radio frequency-generated plasma, a direct current-generated plasma, or a microwave-generated plasma.  
     
     
         55 . The method of  claim 54 , wherein the plasma is an air plasma.  
     
     
         56 . The method of  claim 51 , wherein the mask comprises 96, 384, 1536, or 6144 openings.  
     
     
         57 . The method of  claim 51 , wherein the mask comprises a sacrificial coating positioned proximal to the hydrophobic surface of the solid support.  
     
     
         58 . The method of  claim 51 , wherein contacting the desired locations of the hydrophilic target regions with the plasma further comprises depositing at least 3 W of energy into the plasma.  
     
     
         59 . The method of  claim 51 , wherein contacting with the plasma further comprises generating the plasma using a radio frequency of at least 500 kHz.  
     
     
         60 . The method of  claim 51 , contacting the desired locations of the hydrophilic target regions with the plasma further comprises generating a hydrophilic functionalized polymer comprising carboxyl groups, hydroxyl groups, keto groups, epoxide groups, or a combination thereof.  
     
     
         61 . The method of  claim 60 , wherein the hydrophilic target regions comprise a metal chelating polymer.  
     
     
         62 . The method of  claim 61 , further comprising incubating the hydrophilic target regions with one or more metal ions.  
     
     
         63 . The method of  claim 62 , wherein the one or more metal ions are selected from the group consisting of Fe 3+ , Ga 3+ , Zn 2+ , Ni 2+  and Cu 2+ .  
     
     
         64 . The method of  claim 60 , further comprising incubating the hydrophilic target regions with one or more capture agents capable of associating with the hydrophilic target regions.  
     
     
         65 . The method of  claim 64 , wherein the one or more capture agent comprises an antibody.  
     
     
         66 . The method of  claim 51 , wherein providing the solid support comprises regenerating a previously-used hydrophilic/hydrophobic MALDI support.  
     
     
         67 . The method of  claim 66 , wherein regenerating the previously-used support comprises: 
 contacting one or more surfaces of the support with a plasma and removing organic materials that are attached to the support; surface and    contacting the support surface with a hydrophobic derivatizing agent, thereby forming a hydrophobically-derivatized surface and regenerating the previously-used support.    
     
     
         68 . The method of  claim 67 , further comprising mechanically or chemically stripping the support prior to contacting with the plasma.  
     
     
         69 . The method of  claim 67 , wherein the plasma comprises a radio frequency-generated plasma, a direct current-generated plasma, or a microwave-generated plasma.  
     
     
         70 . The method of  claim 67 , wherein the hydrophobic derivatizing agent comprises an oil or grease film.  
     
     
         71 . The method of  claim 67 , wherein the hydrophobic derivatizing agent comprises one or more of a fluoropolymer and a hydrocarbon polymer.  
     
     
         72 . The method of  claim 71 , wherein the hydrophobic derivatizing agent comprises a 1-4% fluoropolymer solutions in a fluorosolvent.  
     
     
         73 . The MALDI sample support having a hydrophobic surface and one or more hydrophilic target regions as prepared by the method of  claim 51 .  
     
     
         74 . The regenerated MALDI sample support as prepared by the method of  claim 67.